ADP-ribosylation factors (ARFs), a family of 20-kDa guanine nucleotide- binding proteins, were discovered as activators of cholera toxin A subunit (CTA)-catalyzed ADP-ribosylation of the stimulatory GTP-binding protein of the adenylyl cyclase system (Gs alpha) and participate in intracellular vesicular membrane trafficking. ARFs are activated when bound GDP is replaced by GTP and inactivated by hydrolysis of bound GTP to yield ARF-GDP. Usually, ARFs are isolated in an inactive GDP-bound state and require the addition of GTP along with detergent or phospholipid for activity. Purified mutant recombinant ARF1 lacking the first 13 amino acids (rdelta13ARF1-P) stimulated cholera toxin activity essentially equally, with or without added GTP (and phospholipid or detergent). In this study, rdelta13ARF1-P was shown to contain bound nucleotides, which later were identified as GTP and GDP. Nucleotide- free rdelta13ARF1 (rdelta13ARF1-F), prepared by dialysis against 7M urea, was active without added GTP in the absence of SDS, but inactive without added GTP in its presence. Renaturation of rdelta13ARF1-F in the presence of GTP, ITP, GDP or IDP yielded, respectively, rdelta13ARF1-GTP, and rdelta13ARF1-ITP, which were active, and rdelta13ARF1-GDP and rdeltaARF1-IDP, which were inactive. These studies are consistent with the hypothesis that the amino terminus affects nucleotide binding and that an amino-terminally truncated ARF can assume an active conformation in the absence of GTP.